Prostaglandin D2 (PGD2) is formed from the PGH2 cyclooxygenase (COX) product of arachidonic acid by the action of either a lipocalin (L)-like or hemopoietic (H) PGD synthase (Urade et al., 2000, Vitamins and Hormones, 58: 89-120). Both COX enzymes (COX 1 and COX 2) may form PGD2 in vitro, but it is unclear which COX and PGDS enzymes predominate under varied conditions in vivo.
Suppression of PGD2 has been implicated in the bronchoconstriction of aspirin-evoked asthma (O'Sullivan et al., 1996, J Allergy Clin Immunol, 98: 421-432; Bochenek et al., 2003, J Allergy Clin Immunol, 111: 743-749) and release of PGD2 mediates the facial flushing and vascular instability of systemic mastocytosis (Roberts et al., 1980, N Engl J Med., 303: 1400-1404). PGD2 relaxes vascular smooth muscle cells in vitro and its release by dermal dendritic cells contributes to the facial flushing, which complicates administration of the hypolipidemic drug, niacin (Morrow et al., 1989, Prostaglandins, 38: 263-274). PGD2 mediates its effects via activation of D prostanoid receptors (DPs). DP1, a member of the prostanoid family of G protein coupled receptors (GPCRs), mediates the vasorelaxant and bronchodilator effects (Williams et al., 1977, Nature, 270: 530-532; Matsuoka et al., 2000, Science, 287: 2013-2017). DP2, a GPCR of the fMLP receptor subfamily, regulates Th1 and Th2 switching in lymphocytes (Nagata et al., 1999, J Immunol, 162: 1278-1286) and is also expressed on eosinophils and basophils (Nagata et al., 1999, FEBS Lett., 459: 195-199).
Recent interest in PGD2 has been prompted by the use of DP1 blockade as an adjunct to niacin therapy (Cheng et al., 2006, Proc Natl Acad Sci USA, 103: 6682-6687) and by the potential role of PGD2 and its metabolites in the resolution of inflammation (Gilroy et al., 1999, Nat Med, 5: 698-701). However, DP1 is expressed on human platelets and its activation in vitro results in a cyclic-AMP-dependent inhibition of platelet function (Oelz et al., 1977, Prostaglandins, 13: 225-234; Bushfield et al., 1985, Biochem J, 232: 267-271). Nothing is known about the formation of PGD2 or the consequences of its inhibition in hyperlipidemic patients. Aside from a potential role in cardiovascular disease, PGD2 may be of importance in the resolution of inflammation. A potential metabolite of PGD2, 15-deoxy Δ12,14PGJ2, has been postulated to activate PPARγ (Forman et al., 1995, Cell, 83: 803-812) and promote resolution of an inflammatory infiltrate (Gilroy et al., 1999, Nat Med, 5: 698-701). However, it remains to be determined by physicochemical methodology whether formation of 15-deoxy Δ12,14PGJ2 is indeed augmented during the resolution of human inflammation, and although it can activate PPARγ, the concentrations required are unlikely to be attained in vivo (Bell-Parikh et al., 2003, J Clin Invest, 112: 945-955).
Attempts to assess the biosynthesis of PGD2 have been constrained by a paucity of relevant methodology. Aside from asthma and mastocytosis (O'Sullivan et al., 1996, J Allergy Clin Immunol, 98: 421-432; Bochenek et al., 2003, J Allergy Clin Immunol, 111: 743-749; Roberts et al., 1980, N Engl J Med., 303: 1400-1404), little information on biosynthesis of PGD2 in humans has been acquired. Given the evanescence of primary PGs, biosynthesis is classically estimated by measurement of metabolites (McAdam et al., 1999, Proc Natl Acad Sci USA., 96: 272-277; Catella et al., 1986, Proc Natl Acad Sci USA., 83: 5861-5865). However, no metabolites of PGD2 have been reported in mouse, preventing assessment of biosynthetic response to experimental manipulation in that species. Initial attempts at assay development in humans have focused on 11β-PGF2α formed from PGD2 by bovine PGF synthase (Watanabe et al., 1986, Proc Natl Acad Sci USA., 83: 1583-1587) and 2,3-dinor-11β-PGF2α. Both are formed as minor urinary metabolites in monkeys and in a human volunteer following infusion of radiolabelled PGD2 (Liston et al, 1985, J Biol Chem, 260: 13172-13180). However, paired analysis of 11β-PGF2α by gas chromatography mass spectrometry and a commercially available immunoassay revealed poor concordance in the urine of patients with asthma (Misso et al., 2004, Clin Exp Allergy, 34: 624-631; Bochenek et al., 2004, Thorax., 59: 459-464). Quantitative analysis of another major F ring metabolite, 9α,11β-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, has been reported in human plasma and urine (Morrow et al., 1995, J Invest Dermatol, 104: 937-940).
There exists a need in the art for a PGD2 metabolite and assay therefore for use in research methods as well as methods useful for clinical applications. The present invention addresses and meets these needs.